Loss of the p53 signaling pathway, either by mutation or loss of upstream or downstream signaling components, occurs in the vast majority of human cancers. In normal cells, Hdm2 and HdmX coordinately regulate the stability and function of p53. However, each protein is overexpressed in subsets of many different types of human malignancy, leading to inactivation of wild-type p53 function. HdmX overexpression was recently reported to be as high as 20% in breast, colon and lung cancers. Treatment options for tumors harboring wild-type p53 would be greatly expanded if the recent discoveries of low molecular weight compounds capable of disrupting the p53-Hdm2 interaction to induce p53-dependent toxicity continue to show positive results. Preliminary comparison of one small molecule's specificity has shown that HdmX and Hdm2 can be differentially affected, thereby establishing each protein as an independent target for therapy. The goal of this proposal is to develop a new class of small molecules effective against tumor cells retaining wild-type p53 that is transcriptionally repressed due to HdmX overexpression. A cell-based system is proposed that will combine HdmX overexpression with an integrated p53-responsive promoter driving 2- galactosidase expression, an easily measurable and quantifiable specific marker. A screen for compounds that induce p53-dependent transcription will be performed. Candidate compounds that pass through the primary screen will be classified according to their relative strengths and characterized to define the mechanism of p53 activation and the dependence upon HdmX and/or Hdm2 status for efficacy. A limited set of compounds capable of inhibiting HdmX-mediated p53 inactivation will form the basis of a Phase II program aimed at hit-to-lead optimization and the development of prototype therapeutic drugs. PUBLIC HEALTH RELEVANCE: Cancer cells have acquired genetic alterations that disrupt cellular checkpoints established to limit normal proliferation. In many cancers, these checkpoints are simply suppressed rather than absent, leaving the option to reengage and enforce the hidden limits to proliferation as cancer therapy. We propose to identify compounds that can prevent tumors with a common but specific genetic alteration, namely HdmX protein overexpression, from continuing their aberrant growth.